Transmissible spongiform encephalopathies (TSEs or prion diseases) are a group of rare neurodegenerative diseases which include scrapie in sheep, bovine spongiform encephalopathy (BSE), and chronic wasting disease (CWD) in mule deer and elk. In humans, the most common type of prion disease is Creutzfeldt-Jakob disease (CJD) which can occur in several forms. Sporadic CJD (sCJD) makes up the majority of the CJD cases and occurs randomly at an incidence of 1-2 per million people worldwide. Iatrogenic CJD (iCJD) is associated with exposure to prion contaminated medical instruments or products while familial CJD (fCJD) is associated with mutations in the prion protein gene. The infectious agent of TSE diseases is called a prion and is largely composed of an abnormally refolded, protease resistant form (PrPSc) of the normal, protease-sensitive prion protein, PrPC. PrPSc can be deposited in the brain as either diffuse amyloid negative deposits or as dense amyloid positive deposits. For reasons that are not yet clear, amyloid forms of prion disease appear to be less transmissible than non-amyloid forms. Furthermore, it is unknown whether or not prion diseases where PrPSc is deposited primarily as amyloid follow the same pathogenic processes as prion diseases where PrPSc is primarily deposited as non-amyloid. Multiple studies have shown that amyloid formed from amyloid beta (A) protein, alpha synuclein and tau can propagate via a prion-like mechanism and spread from cell-to-cell in transgenic mouse models e.g. Science 313: 1781-1784 (2006), Nat Cell Biol 11: 909-913 (2009), J Exp Med 209: 975-986 (2012). Based on these data, it has been suggested that amyloid formation in neurodegenerative proteinopathies such as Alzheimers Disease (AD) and Parkinsons disease (PD) occur via prion-like mechanisms and that proteins such as AD-associated A may also be transmissible, infectious prions. Co-deposition of misfolded proteins during neurodegeneration, such as the co-localization of PrPSc and A to plaques in some cases of sCJD ACTA Neuropathol 96:116-122 (1998), also suggest that interactions between these proteins could contribute to disease pathogenesis. We are interested in understanding the molecular mechanisms underlying PrP amyloid formation and have begun to approach this issue using both in vitro and in vivo model systems. This project focuses on: 1) Understanding the pathways of PrP amyloid formation and spread and, 2) Studying how mutations and amino acid polymorphisms in PrP influence PrPSc amyloid formation in familial forms of prion disease. Since PrPSc formation and spread appear to be mechanistically similar to the formation and spread of amyloid in other neurodegenerative diseases, the results of our prion studies will likely be broadly applicable to other diseases of protein misfolding and deposition. In 2017, we collaborated with the laboratory of Dr. Jiyan Ma to determine why some self-propagating forms of protease resistant PrP are infectious and others are not. This project was initiated as a direct result of data our laboratory had published examining the relative infectivity of amyloid forms of bacterially derived PrP (Timmes et al. PLoS One 8: e71081 (2013), Annual Report 2013). As suggested by our original PLoS One paper, our collaboration with Dr. Ma has shown that the ability of protease-resistant PrP to self-propagate is not predictive of whether or not it is also infectious. Rather, small structural changes in the N-terminus of self-propagating PrP correlated with infectivity Wang et al. PLoS Pathog. 13: e1006491 (2017). These data have significant implications for the important public health issue of whether or not protein self-propagation via prion-like mechanisms means that diseases such as AD and PD are also infectious. Different proteinase K cleavage sites in the N-terminus of PrPSc are indicative of differences in its structure. Biochemically, the two major PrPSc types associated with CJD, termed Type 1 and Type 2, can be distinguished by the molecular mass of PrPSc following protease digestion suggesting that there are two major structural isoforms of PrPSc PNAS 97: 10168 (2000). Type 1 PrPSc has an N-terminus starting primarily at amino acid residue 82 and a protease-resistant molecular mass of approximately 21 kDa. Type 2 PrPSc has an N-terminus starting primarily at amino acid residue 97 and a protease-resistant molecular mass of approximately 19 kDa. However, it is difficult to explain the variable phenotypes seen in CJD in the context of just two structural PrPSc isoforms. Recently, it has been found that many cases of sCJD are mixtures of Type 1 and Type 2 PrPSc suggesting that there may be a complex population of PrPSc molecules present with different secondary structures Brain 132: 2643 (2009). In 2017, we initiated a project to use LC-MS/MS Nanospray Ion Trap Mass Spectrometry to precisely map the N-termini of PrPSc molecules associated with different neurological subtypes of CJD. The goal of this project is to determine whether certain structural populations of PrPSc correlate with specific CJD phenotypes. In 2017, we continued our long term in vivo work studying the pathogenesis of different forms of amyloid and non-amyloid human prion disease in transgenic mice expressing wild-type human PrP. Multiple CJD isolates were inoculated into these mice and, in some cases, multiple brain regions from the same patient were inoculated. We are currently in the process of completing the complex analysis of the neuropathology and deposition of PrPSc in the brains of these mice as well as the biochemical analysis of the PrPSc present in the brain. This experiment represents another approach in delineating the mechanisms underlying amyloid and non-amyloid forms of prion disease. These studies will provide important insight into several poorly understood areas of human prion disease including 1) the contribution of the host versus the contribution of the prion strain to different in vivo disease phenotypes and, 2) the mechanisms of amyloid versus non-amyloid prion formation. Finally, in 2017 we continued a collaboration with Dr. Pedro Piccardo using mass spectrometry to study BSE-infected non-human primates (NHP). These animals develop a neurodegenerative disease characterized by accumulation of PrPSc, hyper-phosphorylated tau, and alpha synuclein J Gen Virol 95:1612-16-18 (2014). Over the last year, this project has required significant methods development. In 2017, we successfully determined the best protocol to obtain the greatest number of high-resolution protein identifications from our samples. We have now begun to apply this technique to the BSE-infected NHP brain samples from Dr. Piccardo and are in the process of analyzing the proteomics data obtained. This experimental model will enable us to better understand the molecular mechanisms behind neurodegeneration in complex proteinopathies.